Prevention of autoimmune diseases by aerosol administration of autoantigens

ABSTRACT

Disclosed herein is a method for treating autoimmune diseases in mammals by administration of one or more agents selected from the group consisting of autoantigens specific for the autoimmune disease, disease-suppressive fragments and analogs of said autoantigen in aerosol form.

This is a continuation of application Ser. No. 08/419,502, filed Apr.10, 1995 pending; which is a continuation of application Ser. No.08/053,306, filed Apr. 26, 1993 now abandoned; which is a continuationof application Ser. No. 07/454,806, filed Dec. 20, 1989 now abandoned;which is a continuation-in-part of application Ser. No. 07/379,778 filedJul. 14, 1989 now abandoned; which is a continuation-in-part ofPCT/US88/0213, filed Jun. 24, 1988; which is a continuation-in-part ofapplication Ser. No. 07/065,734, filed Jun. 24, 1987, now abandoned.

BACKGROUND OF THE INVENTION

This invention pertains to an improvement in the treatment of autoimmunediseases. More specifically, the invention is directed to theadministration of autoantigens and biologically active fragments oranalogs of such autoantigens in aerosol form for the prevention andtherapeutic treatment of autoimmune diseases. The invention alsoincludes aerosol formulations of autoantigens useful in the treatment ofautoimmune diseases in mammals.

Autoimmune diseases are characterized by an abnormal immune responseinvolving either cells or antibodies, that are in either case directedagainst normal autologous tissues.

Autoimmune diseases in mammals can generally be classified in one of twodifferent categories: cell-mediated disease (i.e. T-cell) orantibody-mediated disorders. Non-limiting examples of cell-mediatedautoimmune diseases include multiple sclerosis, rheumatoid arthritis,autoimmune thyroiditis, diabetes mellitus (Juvenile onset diabetes) andautoimmune uveoretinitis. Antibody-mediated autoimmune disorders includemyasthenia gravis and systemic lupus erythematosus (or SLE).

The current treatments for both categories of autoimmune diseasesinvolve administration of drugs which non-specifically suppress theimmune response. Examples of such drugs are methotrexate,cyclophosphamide, Imuran (azathioprine) and cyclosporin A. Steroidcompound such as prednisone and methylprednisilone are also employed inmany instances. These drugs have limited efficacy against both cell- andantibody-mediated autoimmune diseases. Use of such drugs is limited byvirtue of their toxic side effects and also because they induce "global"immunosuppression in a patient receiving prolonged treatment with thedrug, e.g. the normal protective immune response to pathogenicmicroorganisms is downregulated thereby increasing the risk ofinfections caused by these pathogens. A further drawback is that thereis an increased risk that malignancies will develop in patientsreceiving prolonged global immunosuppression.

Other therapies have been proposed for the treatment of autoimmunediseases. U.S. patent application Ser. No. 65,794 filed Jun. 24, 1987(now abandoned) and copending International Patent ApplicationPCT/US88/02139 filed Jun. 24, 1988, disclose that the oral or enteraladministration of relatively high levels of myelin basic protein (MBP)and disease-inducing and non-inducing fragments and analogs thereof iseffective in suppressing acute monophasic experimental allergicencephalomyelitis (EAE), an induced T-cell-mediated autoimmune diseasedirected against myelin basic protein. EAE is a recognized and widelyused animal model for multiple sclerosis (MS). The above-identifiedapplications also disclose that the oral or enteral administration ofMycobacterium tuberculosis is an effective treatment for suppressingadjuvant arthritis and extrapolate the aforementioned results to thetreatment of other autoimmune diseases.

Copending U.S. patent application Ser. No. 379,778 filed Jul. 14, 1989discloses the oral or enteral administration of S-antigen for thetreatment of autoimmune uveoretinitis.

Various methods have been employed to induce antigen-specificsuppression of EAE such as immunization with MBP emulsified inincomplete Freund's adjuvant (Lando, Z. et al., J.P. Immunol., 126:1526, 1981), or an intravenous injection of MBP coupled to lymphoidcells (Sriram, et al., Cell Immunol. 75: 378, 1983).

Traugott et al., J. Neurol. Sci. 56: 65-73, 1982 and Raine et al., Lab.Investigation 48: 275-84, 1983 teach that treatment of a strain ofguinea pigs suffering from chronic relapsing EAE by parenteraladministration of MBP alone or, in incomplete Freund's adjuvant (IFA)or, in combination with galactocerebroside, a lipid hapten of myelin,suppressed the clinical symptoms of EAE.

Based on an English language abstract, Belik et al., Vopr. Mev. Khin.24: 372-377, 1978, disclose the parenteral administration of "alkalinemyelin protein fragment (AMPF)" and "synthetic encephalitogenic peptide(SEP)" to guinea pigs with EAE. The animals had been sensitized bybovine AMPF or synthetic SEP but recovered after administration of AMPF.

Braley-Mullen et al., Cell Immun. 51: 408, 1980, disclose thesuppression of the symptoms of experimental autoimmune thyroiditis inguinea pigs by injection of thyroglobulin antigen in IFA.

Nagler-Anderson et al., Proc. Natl. Acad. Sci. USA 83: 7443-7446, 1986disclose the oral administration of collagen to suppresscollagen-induced arthritis in a mouse model. Type II collagen-inducedarthritis was suppressed in the mouse by intragastric administration ofsoluble, but not denatured, Type II collagen prior to immunization ofthe animal with Type II collagen in an adjuvant. The Nagler-Andersonantigen was not administered in aerosol form.

Other investigators have examined the effects of aerosolized proteins onthe treatment of genetic diseases and on modulating the IgE response inmammals.

Hubbard, R. C. et al. (Proc. Natl. Acad. Sci. (USA) 86: 680-684, 1989)demonstrated the feasibility of administered proteins to mammals inaerosol form. Hubbard et al. disclose administration of a relativelylarge protein alpha₁ -antitrypsin (AAt) via the pulmonary epithelialsurface for the treatment of alpha anti-trypsin deficiency, an inheritedgenetic disease. AAt, a 45,000 dalton molecular weight single-chainpolypeptide (that functions as an inhibitor of neutrophil elastase) wasadministered to sheep in an aerosol form. Aerosolized AAt remained fullyfunctional and intact in the tissues of the mammal to which the materialwas administered and diffused across the alveolar epithelium, asevidenced by the presence of AAt in the lung, lymph and blood tissue.

Holt, P. G. et al., Immunol. 42: 409-417, 1981 disclose the inhibitionof specific IgE responses in mice by pre-exposure to inhaled antigens.The exposure of mice to aerosolized ovalbumin once weekly for sevenweeks caused the suppression of IgE responses when these animals werechallenged intraperitoneally with Soluble or alum-precipitatedovalbumin.

Sedgwick, J. D. et al., Immunol. 56: 635-642, 1985 reported on thedown-regulation of the specific IgE response to an inhaled antigen.Repeated exposure of rats to an aerosol of ovalbumin inducedcarrier-specific tolerance to subsequent challenge with the sameantigen.

Holt, E. G. et al., Immunol. 60: 97-102, 1987 disclose that repeatedinhalation of low levels of ovalbumin by mice or rats preferentiallyinduced tolerance to the IgE antibody class and postulated that thisprocess represented an important protective mechanism that normallyprevents allergic sensitization of a mammal to air-borne antigens.

Sedgwick, J. D. et al., Cell Immunol. 94: 182-194, 1985 disclose thatthe repeated exposure of high-IgE-responder rats to an aerosol of anantigen once weekly triggered progressively increasing levels ofantigen-specific IgG in the serum. Adoptive transfer of splenocytes fromaerosol-exposed animals to naive rats caused a suppression of IgEresponses without affecting specific IgG responses.

No method or technique for treating or preventing autoimmune diseases issuggested or disclosed in these references. It is an object of thepresent invention to provide methods for treating mammals suffering fromautoimmune diseases.

Another object of the present invention is to provide pharmaceuticalformulations for treating mammals suffering from autoimmune diseases.

A still further object of the invention is to provide a formulation foradministration to mammals for the purpose of preventing or treatingautoimmune diseases in such mammals.

These and other objects of the present invention will be apparent tothose of ordinary skill in the art in light of the presentspecification, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the disease suppressive effects of variousconcentrations of orally administered MBP on the course and severity ofEAE;

FIG. 2 is a graph showing the disease suppressive effects of variousconcentrations of MBP administered in aerosol form on the course andseverity of EAE;

FIG. 3(A-D) are a series of graphs directly comparing the effects ofvarious doses of GP-MBP when administered orally or in aerosol form onthe course and severity of EAE.

FIG. 4 is a graph showing the effects of GP-MBP fragments on the courseand severity of EAE.

FIG. 5 is a graph showing treatment of animals after induction of EAE.

FIG. 6 is a graph showing the effects of various aerosol-administeredproteins on EAE.

FIG. 7 is a graph showing the effects of aerosolized collagen on thecourse and severity of collagen-induced arthritis.

FIG. 8 is a graph showing the transfer of immunity against EAE torecipient animals mediated by spleen cells isolated from animals treatedaccording to the method of the present invention.

SUMMARY OF THE INVENTION

It has now been discovered that an improved and more effective methodfor preventing or treating autoimmune diseases in mammals comprisesadministration in aerosol form of one or more autoantigens specific forthe autoimmune disease to be prevented or treated in said mammal.

In one aspect, the present invention provides a method for treating orpreventing an autoimmune disease in a mammal by administering to suchmammal an effective amount of an aerosol composition comprising at leastone autoimmune suppressive agent selected from the group consisting ofautoantigens that are specific for such autoimmune disease, autoimmunesuppressive fragments and analogs of such autoantigens.

In another aspect, the invention embraces aerosol dosage forms for usein treating autoimmune diseases in meals. The dosage forms comprise atleast one autoimmune suppressive agent selected from the groupconsisting of autoantigens specific for said autoimmune disease,autoimmune suppressive fragments and analogs of said autoantigens inaerosol form.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references referred toin this specification are hereby incorporated by reference in theirentirety.

It has now been discovered that administration of autoantigens (orautoimmune suppressive fragments or analogs thereof) in aerosol form iseffective in treating autoimmune disease in mammals. A particularlysurprising and unexpected development is the discovery thatadministration of autoantigens in aerosol form is more effective inpreventing and treating autoimmune diseases in mammals thanadministration of the same autoantigens in solid form via the oralroute. Also surprising is the discovery that it is possible to achieveeffective suppression and prevention of autoimmune diseases in mammalsusing a smaller quantity of such autoantigens in an aerosol form than byadministration of a solid dosage form. The aerosol administration ofautoantigens has been found to be effective in suppressing bothcell-mediated and antibody-mediated autoimmune responses.

Non-limiting examples of autoimmune diseases that may be treated orprevented using the method of the present invention include multiplesclerosis, rheumatoid arthritis, myasthenia gravis, autoimmunethyroiditis, diabetes mellitus (especially Juvenile Onset Diabetes),autoimmune uveoretinitis, systemic lupus erythematosus (SLE or Lupus),adrenalitis and chronic active hepatitis.

As used herein, the term "aerosol" refers to finely divided solid orliquid particles that may be created using a pressurized system such asa nebulizer. The liquid or solid source material contains autoantigensand/or autoimmune disease suppressive fragments and analogs thereof asdefined herein.

An autoimmune disease is a malfunction of the immune system of mammals,including humans. In a mammal afflicted with such a disease, the immunesystem cannot or does not distinguish between exogenous (foreign)substances within the mammal and autologous tissues or substances. As aresult, the immune system treats autologous tissues (self antigens) andsubstances as if they were foreign and evokes the proliferative immunedefense that is usually reserved for use against exogenous (foreign)tissues or invading organisms. In essence, one arm of the normal immunesystem becomes altered and begins a proliferative response againstautologous tissues. As employed herein, the term "mammal" refers to alllife forms that have an immunoregulatory system and are thereforesusceptible to autoimmune diseases.

As employed herein, the term "autoantigen" refers to any substancenormally found within a mammal that (1) is not recognized as part of themammal itself by the lymphocytes or antibodies of that mammal, (2) isattacked by the immunoregulatory system of the mammal as though suchantigen were a foreign substance and (3) acts to downregulate the arm ofthe immune system that is responsible for causing a specific autoimmunedisease. The term autoantigen also includes antigenic substances whichinduce conditions having the symptoms of an autoimmune disease whenadministered to mammals.

As used herein the term "autoimmune suppressive fragments" includes anypeptide or polypeptide containing partial amino acid sequences ormoieties of autoantigens and possessing the ability to suppress orprevent an autoimmune response upon aerosol administration. Suchfragments need not possess the autoantigenic properties of the entireautoantigen. By way of non-limiting example, when MBP is administeredparenterally to meals in the presence of an adjuvant it induces EAE insusceptible mammals. It has now been discovered that certainnon-disease-inducing fragments of MBP (i.e., fragments of MBP which donot induce EAE when administered parenterally with an adjuvant)nevertheless possess autoimmune-suppressive activity when administeredorally (or enterally) or in aerosol form to mammals suffering from EAE.Examples of such fragments are reported in U.S. patent application Ser.No. 65,734, and International Patent Application No. PCT/US88/02139, andExamples 2 and 6 below.

As employed herein the term "analogs" of such autoantigens or fragmentsthereof refers to compounds that are structurally related to theseautoantigens or to their autoimmune-suppressive fragments and whichpossess the same biological activity, i.e. the ability to eliminate orsuppress the autoimmune response, upon aerosol administration. By way ofnon-limiting example, the term includes peptides having amino acidsequences which differ from the amino acid sequence of the autoantigenor disease suppressive fragments thereof by one or more amino acidresidues (while still retaining the autoimmune-suppressive activity ofthe autoantigen or fragment) as well as compounds or compositions whichmimic the autoimmune-suppressive activity of the autoantigen in itsability to suppress or alleviate the symptoms of the disease. Oneexample is tissue from an organ that is the target of attack by an armof the immune system in an autoimmune disease, e.g. the pancreas indiabetes or the white matter of the central nervous system in multiplesclerosis. Another exemplary analog is peptide S79 as disclosed incopending U.S. patent application Ser. No. 65,734 filed Jun. 24, 1987.

As used herein the term "autoimmune-disease suppressive agent" or"autoimmune suppressive agent" refers to a compound or composition whichcan be administered in an aerosol form to a mammal to suppress, preventor delay the clinical onset or manifestation of a specific autoimmunedisease. The term includes autoantigens that are active against aspecific autoimmune disease, as well as autoimmune-suppressive fragmentsor analogs thereof as defined above.

As employed herein the term "treatment" refers to prophylacticadministration to prevent an autoimmune disease in susceptibleindividuals or to treatment of an active autoimmune disease in anaffected individual.

The tolerance induced by the autoimmune-suppressive agents of thisinvention is dose-dependent; over a broad dosage range of aerosolmaterial it has been found that suppression (or attenuation) of clinicalmanifestations of the disease (EAE) increases with increasing dosagelevels of the aerosolized autoimmune-suppressive agent administered asshown in Examples 2-9 below. Dose dependency was also seen in thearthritis system. Moreover, the aerosol administration of an irrelevantantigen (i.e. one not implicated in an autoimmune disease, such ashistone protein, or certain synthetic fragments of MBP) has no effect onthe clinical manifestation of the autoimmune disease.

Administration of autoantigens and the aerosol route for the treatmentof autoimmune disease has several advantages over other routes ofadministration. Ease of administration is one important advantage. Also,as shown below in Example 2, aerosol administration of MBP is effectivein treating EAE at substantially lower doses than those required totreat this disease when the same agent was administered orally via theoral route in a solid dosage form. A further advantage is that theaerosol administration route involves less exposure of the autoimmugenicagents of the present invention to degradative gastric juices, which mayact to reduce the efficacy of such agents.

It should be noted that the amount of autoimmune suppressive agent ofthe present invention which the treated animal receive via aerosoladministration is substantially lower than the total amount of the agentwhich is administered. It is believed that only 1/200 of the totaldosage present in the nebulizer is actually taken up on the pulmonarysurface of the treated animals. The majority of the autoimmunesuppressive agent nebulized into the cages is not breathed by theanimals but non-specifically adheres to the cages and to the animals.Therefore, aerosol administration is much more effective than oral orenteral administration where in the latter case all of the autoimmunesuppressive agent is delivered to the treated animals.

Various model systems have been developed for studying autoimmunediseases. Experimental allergic encephalomyelitis (EAE) is an inducedT-cell mediated autoimmune disease which has been studied in mice andother mammalian species as a model for Multiple Sclerosis (MS) inseveral mammalian species. The disease is induced by parenteraladministration of MBP and an adjuvant (such as Freund's completeadjuvant). This treatment induces both a monophasic and anexacerbating/remitting form of demyelinating disease (depending on thespecies and details of administration). The induced disease has thecharacteristics of MS. Parenteral administration of Mycobacteriumtuberculosis with Freund's complete adjuvant oil into the dorsal roottail of susceptible mammals induces a disease with the characteristicsof human rheumatoid arthritis. In addition, the administration to Lewisrats of S-antigen and an adjuvant induces autoimmune uveoretinitis.Diabetes develops spontaneously in the NOD mouse and the BB rat. Variousones of these model systems have been employed to demonstrate theefficacy and improved treatment provided by the present invention.

The present invention may be used to treat a wide variety of autoimmunediseases, both antibody- and cell-mediated. As shown below in Examples 6and 7, aerosol administration of an autoantigen (guinea pig MBP) anddisease-suppressive fragments thereof caused suppression of bothcell--(e.g. delayed-type hypersensitivity reactions) andantibody-mediated immune responses. In addition, as shown in the datapresented in Example 3, this suppression was actively mediated by spleencells, implying a role for active cellular suppression in thisphenomenon. Therefore, as in the case of immune suppression mediated bythe oral administration of autoantigens (disclosed in U.S. patentapplication Ser. No. 65,734 and the copending International PatentApplication PCT/US88/02139), aerosol administration of autoantigens isbelieved to act, at least in part, by mediating the activity ofsuppressor T-cells. Non-limiting examples of autoimmune diseases whichare cell-mediated include multiple sclerosis, rheumatoid arthritis,autoimmune uveoretinitis, diabetes and autoimmune thyroiditis.Antibody-mediated autoimmune diseases include myasthenia gravis,systemic lupus erythematosus (SLE), pemphigus and thrombicthrombocytopenic purpuria. A non-limiting list of disease models and thespecific autoantigens effective in the treatment of these diseases whenadministered in an aerosol form are set forth below in Table 1.

                  TABLE 1                                                         ______________________________________                                        Disease Model    Specific Autoantigen                                         ______________________________________                                        Multiple Sclerosis                                                                             MBP                                                          Rheumatoid Arthritis                                                                           Collagen                                                     Autoimmune Thyroiditis                                                                         Thyroglobulin                                                Myasthenia Gravis                                                                              Acetylcholine receptor                                       Autoimmune uveoretinitis                                                                       S-antigen                                                    Systemic Lupus Erythematosus                                                                   DNA                                                          Diabetes         Islet cell extract                                           Chronic Active Hepatitis                                                                       Liver extract                                                Adrenalitis      Adrenal gland extract                                        Polymyositis     Muscle extract                                               Autoimmune hemolytic anemia                                                                    Hematopoietic cells                                          Rheumatic carditis                                                                             Heart extract                                                Scleroderma      Skin cell extract                                            ______________________________________                                    

For any auto immunes disease, tissue extracts can be used as well as thespecific antigens described above.

Other autoimmune diseases and their specific autoantigens and/or targettissues are disclosed in Schwartz, R. S. et al. in FundamentalImmunology, Second Edition, Paul, W. E., Ed., pg 819-859, Raven Press,NY, 1989.

Autoantigens for use in the present invention can be isolated from thetissue which is the target for the particular autoimmune disease. Forexample, myelin basic protein (MBP) for use in treating MS can beisolated and purified from meals using the method of Diebler et al.(infra) as shown in Example 1 below.

When treating a disease having the symptoms of rheumatoid arthritis,collagen can be isolated and purified by the method of Trentham et al.,J. Exp. Med. 146: 857, 1977.

For treating autoimmune uveoretinitis, purified S-antigen can beobtained as described in copending application Ser. No. 379,778.

For treating myasthenia gravis, purified acetylcholine receptor can beisolated by the method of Mcintosh et al. J. Neuroimmunol. 25: 75, 1989.

Fragments and analogs of autoantigens for use in the present inventioncan be synthesized using solid phase synthesis techniques well-known inthe art such as those of Merrifield, R. B. (Fed. Proc. Am. Soc. Ex.Biol. 21: 412, 1962 and J. Am. Chem. Soc. 85: 2149, 1963) and Mitchel,A. R. et al.) as well as Tam, J. et al, (J. Am. Chem. Soc. 98: 7357,1976). Analogs can be constructed by identifying an equivalent aminoacid sequence and using the peptide synthesis techniques disclosedabove.

Analogs can be provided using the known amino acid sequence of GP-MBP asdisclosed in G. Hashim, in Meylin: Chemistry and Biology Alan R. Lisa,N.Y., 1980 using techniques described above and in Eyler, E. H., inAdvances in Experimental Medicine and Biology 98: 259-281, 1978. Forexample, a peptide having a sequence corresponding to GP-MBP amino acidresidues 72-85 as disclosed in Hashim (supra) can be chemicallysynthesized using the above-described technique with an amino acidsubstitution at the terminal asparagine position to glutamine. Thepeptide can be tested for disease-suppressive activity when administeredin aerosol form using the techniques as shown in Example 2 below.

Disease-suppressive analogs and fragments can also be obtained usingrecombinant DNA techniques well-known in the art.

The present invention also provides aerosol pharmaceutical formulationsand dosage forms for use in treating mammals suffering from autoimmunediseases. In general such dosage forms contain one or more autoimmunesuppressive agents selected from the group consisting of autoantigensdirected against the autoimmune disease, disease suppressive fragmentsand analogs of such autoantigens, in an amount effective to treat orprevent the clinical symptoms of the specific autoimmune disease. Anystatistically significant attenuation of one or more symptoms of anautoimmune disease that has been treated pursuant to the method of thepresent invention is considered to be a treatment of such disease withinthe scope of the invention.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual aerosol dose of each dosage formneed not in itself constitute an effective amount for treating theparticular autoimmune disease since the necessary effective amount canbe reached by administration of a plurality of dosage units.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.

The route of administration of the suppressive agents of the presentinvention is in an aerosol or inhaled form. The suppressive agents ofthe present invention can be administered as a dry powder or in anaqueous solution. Preferred aerosol pharmaceutical formulations maycomprise for example, a physiologically-acceptable buffered salinesolution containing between about 0.15 mg and about 300 mg of one ormore of the autoimmune-suppressive agents of the present inventionspecific for the autoimmune disease to be treated.

Dry aerosol in the form of finely divided solid autoantigen particlesthat are not dissolved or suspended in a liquid are also useful in thepractice of the present invention. Autoantigen may be in the form ofdusting powders and comprise finely divided particles having an averageparticle size of between about 1 and 5 um, preferably between 2 and 3um. Finely divided autoantigen particles may be prepared bypulverization and screen filtration using technique well known in theart. The particles may be administered by inhaling a predeterminedquantity of the finely divided material, which can be in the form of apowder.

Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically-acceptable buffered saline solutionssuch as phosphate buffered saline solutions pH 7.0-8.0.

In general, the autoantigen, fragment or analog is introduced to amammal in an aerosol form in an amount ranging between about 0.1 mg perkg body weight of said mammal and about 15 mg per kg body weight of saidmammal per day, and may be administered in a single dosage form ormultiple dosage forms. Preferably, the autoantigen, fragment or analogis administered in an amount ranging between 1 mg and about 10 mg per kgbody weight of said mammal per day. The exact amount to be administeredto a patient will vary depending on the stage and severity of thepatient's disease and the physical condition of the patient.

The pharmaceutical formulations of the present invention may beadministered in the form of an aerosol spray using for example, anebulizer such as those described in U.S. Pat. No. 4,624,251 issued Nov.25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No.3,561,444 issued Feb. 9, 1971 and U.S. Pat. No. 4,635,627 issued Jan.13, 1971. The aerosol material is inhaled by the subject to be treated.In the present examples (and for purposes of accuracy) the animalstreated with aerosol agents were retained in enclosed (airtight) cages,into which the aerosol was dispensed. Thus, the amount of material perunit of area could be determined and the results quantified in terms ofunit of aerosol material per unit volume of cage area.

Other systems of aerosol delivery, such as the pressurized metered doseinhaler (MDI) and the dry powder inhaler as disclosed in Newman, S. P.in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds. pp. 197-224,Butterworths, London, England, 1984, can be used when practicing thepresent invention.

Aerosol delivery systems of the type disclosed herein are available fromnumerous commercial sources including Fisons Corporation (Bedford,Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co.,(Valencia, Calif.).

The working examples presented below illustrate that administration ofMBP and Type II collagen in aerosol form was effective in suppressingthe clinical symptoms of EAE and collagen-induced arthritis,respectively in a dose-dependent fashion over a wide dosage range.Administration of MBP in aerosol (liquid) form was effective insuppressing EAE when administered before or after disease induction andresulted in both a delay in the onset of disease symptoms and areduction in the disease severity in treated animals. Both specificantibody and cell-mediated (e.g. delayed-hypersensitivity) reactionswere downregulated by aerosol administration of specific autoantigens ordisease suppressive fragments thereof. In these instances the specificimmunosuppression induced by aerosol administration of autoantigen wasmediated by spleen cells and not cells isolated from the thymus oftreated animals. These spleen cells were able to actively transferprotection against EAE to naive recipients. Moreover, bovine myelin wasalso found to be effective in suppressing EAE as was guinea pig MBP,although the latter was more effective disease suppressive agent in ratsthat had been immunized with GP-MBP to induce EAE. Finally, histologicalexamination of aerosol-treated animals showed that there was an absenceof cells actively infiltrating the brains of animals treated accordingto the present invention. Thus, aerosol administration of an autoantigento animals suffering from an autoimmune disease was shown to affectclinical symptoms of the disease and prevented cells from migrating intothe brain of treated animals. The latter is believed to be a possiblemechanism of demyelination causing the symptoms of EAE.

Further details of the invention are set forth below in workingexamples.

EXAMPLE 1

Autoantigens used in practicing the method of the present invention wereobtained using the method and techniques set forth below.

GP-MBP was purified by the method of Diebler, G. E. et al. (Prep.Biochem. 2: 139, 1972) from guinea pig brain tissue and was obtainedfrom Pel Freeze (Rogers, Ark.). Briefly, central nervous system tissuewas isolated and homogenized in a chloroform-methanol solution,extracted in acetone, filtered and resuspended in the same solution. Thesolution was extracted in acetone, filtered, resuspended in water,adjusted to pH 3.0 and incubated for 1 hour. The solution was thencentrifuged and extracted with 8M urea, CM-5 was added, the pH adjustedto 11, the solution filtered and resuspended twice in urea. The solutionwas then filtered again, resuspended twice in water, filtered,resuspended in 0.121N HCl, filtered, dialyzed against 10 volumes ofdistilled water and lyophilized before use.

Type II collagen used in the Examples presented below was purchased fromGenzyme (Boston, Mass.).

EXAMPLE 2

In the experiments described below, Lewis rats, age 6-8 weeks (CharlesRiver, Wilmington, Mass.) in which EAE had been induced on day 0 byfootpad injection of 10 micrograms of guinea pig myelin basic protein(GP-MBP) purified as in Example 1 above, in Freund's complete adjuvantas disclosed in J. Immunol. 140: 440, 1988. The rats were treated asdescribed below with various doses of. GP-MBP in concentrations rangingbetween 0.005 milligrams and 5 milligrams either orally or in an aerosolspray.

The oral and aerosolized GP-MBP were administered in the same doses onthe same days to groups of five rats.

The aerosol GP-MBP was administered in phosphate buffered saline (PBS,pH 7.4) using a nebulizer. Aerosol was administered to test animalsthrough a hole punched in the side of the cage which held the animals.For aerosolization, a nebulizer (American Pharmoseal Co., Valencia,Calif., Catalog No. 002038) was attached to an air pressure outletdelivering the equivalent of 7.4 liters of oxygen (the amount of oxygenused in a hospital for nebulization). The nebulizer produced droplets ofspray having a diameter of between about 0.3 micrometers and about 0.5micrometers in diameter. 25 mg of GP-MBP, purified according to themethod of Diebler et al. (supra) was dissolved in 5 ml of PBS. This wasthen aerosolized over a 10 to 15 minute period to 5 rats per cage(having dimensions 14"×12"×7", for height, width and depth,respectively). During aerosolization, a fine mist was created in thecage and the rats moved about freely. The schedule of treatments was asfollows: Treatments (either oral or aerosol) were given on days -10, -7,-5, -3, 0, +2 and +4. Immunization to induce EAE on day 0 resulted in anacute paralytic disease with symptoms manifesting on days 12 to 14post-immunization. The scoring system was: 0=normal; 1=loss of tailtone; 2=weakness of back legs; 3=paralysis of back legs; 4=front legweakness; and 5 =moribund. EAE severity varied between 2 and 5,depending on the experiment. Animals were scored clinically in a blindedfashion.

Oral GP-MBP was administered as disclosed in copending InternationalApplication No. PCT/US88/02139 and U.S. patent application Ser. No.65,734.

The results are shown in FIGS. 1 and 2.

The graph in FIG. 1 illustrates that the control animals beganmanifesting disease symptoms at approximately 12 days after induction ofEAE. The oral administration of GP-MBP caused a significant decrease indisease symptoms and delayed the onset of disease symptoms in all groupstested with the maximal delaying effects occurring when 0.5 mg or 5 mgof GP-MBP was administered orally per animal.

In FIG. 2 it can be seen that the aerosol administration of from 0.005mg to 5 mg of GP-MBP significantly decreased EAE disease severity in allthe animals tested. Administration of 5 mg of GP-MBP totally protectedall animals by suppressing all disease symptoms. Aerosol administrationwas more effective than oral administration, i.e. administration ofGP-MBP in aerosol (spray) form reduced disease symptoms at lowereffective concentrations (dosage levels) than administration of GP-MBPin solid form via the oral route.

A direct comparison of each dose (i.e. 0.005, 0.05, 0.5 and 5mg/animal), administered either orally or in aerosol form is shown inFIGS. 3A-D. The experiments were performed exactly as above using 5animals per experimental group. The results confirmed thataerosolization was more effective than oral administration in thatsmaller quantities of aerosolized GP-MBP were required to suppress EAEdisease symptoms This data shows that the route of administration of thematerial inhaled in the lungs caused the effects shown in FIGS. 1-3 andwas not simply due to material that might be swallowed because muchlarger quantities of orally-administered GP-MBP are required to producethe same effect as when administered in aerosol form.

Different fragments of GP-MBP were tested for their effects on EAE. TheMBP fragments were prepared by solid phase peptide synthesis (obtainedfrom Biosearch, San Raphael, Calif.). Fragments corresponding to aminoacid residues 16-35, 51-70 and 121-138 of GP-MBP were administered to 5animals at a concentration of 0.14 mg per animal as described above.None of the above administered fragments were encephalotgenic (i.e. noneof the fragments induce EAE upon parenteral administration with anadjuvant). The data is shown in FIG. 4.

In FIG. 4, fragments corresponding to amino acid residues 16-35 and51-70 of GP-MBP were effective in suppressing the disease symptoms ofEAE. A fragment corresponding to amino acid residues 121-138 of GP-MBPhad no protective effect on EAE in the treated animals and was similarto controls.

In order to show that aerosolization of GP-MBP was effective in treatingEAE when administered after induction of the autoimmune disease, animalswere treated with 5 mg GP-MBP per animal either 3 days before inductionof EAE or on days +7, +9 and +11 after disease induction. The resultsare shown in FIG. 5.

The data in FIG. 5 illustrate that aerosol administration of GP-MBP waseffective in suppressing Whether given 3 days prior to or on days +7,+9, +11 post-induction of EAE.

In order to show the specificity of the autoimmune suppressivetreatment, different concentrations of GP-MBP, a fragment of GP-MBP(corresponding to amino acid residues 21-40 of GP-MBP) or histoneprotein (5 mg/animal, obtained from Sigma Chemical Co., St. Louis, Mo.)were aerosolized to rats in which EAE had been previously induced. Theresults are shown in FIG. 6.

The data in FIG. 6 show that when 5 mg of GP-MBP was administered inaerosol form, it completely protected the treated animals from inductionof EAE. One mg of GP-MBP administered in aerosol form also wasprotective. Histone protein at 5 mg did not protect against EAE. Histonewas used as a control because it is a positively charged protein (as isMBP) and is of a similar molecular weight to MBP, (18,000 daltons ascompared to 16,000 daltons for MBP).

In this experiment the GP-MBP fragment administered (21-40) also did notprotect. In FIG. 6 it can be seen that 1 mg of GP-MBP given orallyprotected the animals against EAE but did not protect as well as whenthe same amount of GP-MBP was given in aerosol form.

EXAMPLE 3

The data presented below represent a series of in vitro experimentswhich illustrate that spleen cells recovered from animals treated withaerosolized GP-MBP actively suppressed proliferative responses in vitro.Animals received autoantigen in aerosol form on six separate occasionsusing the identical regimen as in Example 2 above except that they didnot receive the -10 day treatment. Modulator cells included thymocytesor splenocytes from treated or non-treated animals. Responder cellsincluded NLB, which is a myelin basic protein specific T-cell linederived from Lewis rats, and NLA is a mycobacterial induced line derivedfrom Lewis rats, the latter used as a negative control. The NLB and NLAcell lines were derived from the popliteal lymph nodes of Lewis ratsimmunized with either GP-MBP or Mycobacterium tuberculosis, respectivelyin Freund's complete adjuvant using techniques well-known in the art (J.Immunol. 131: 2810, 1988). The isolated lymph node cells wererestimulated in vitro with antigen plus interleukin-2 (IL-2) before use.The experiments were performed as described in Lider et, al, (J.Immunol. 142: 748-752, 1988). The amount of MBP or Concanavalin A (ConA) used in vitro was 10 micrograms/ml and 1.25 micrograms/ml,respectively. Con A was used as a non-specific control. The results areset forth in Tables 2 and 3 below.

                                      TABLE 2                                     __________________________________________________________________________    SUPPRESSION OF LINE CELLS USING                                               THYMOCYTES FROM TREATED RATS AS MODULATORS                                             CONTROL RATS                                                                            AEROSOL TREATED RATS                                                                          %                                          RESPONDERS                                                                             (Δ CMP × 10.sup.3)                                                          (Δ CMP × 10.sup.3)                                                                SUPPRESSION                                __________________________________________________________________________    NLB + CON A                                                                            24.648 ± 0.9                                                                         29.950 ± 6.6 -17%                                       NLB + GP-MBP                                                                           20.647 ± 6.9                                                                         22.480 ± 5.6 -8%                                        NLA + CON A                                                                            19.690 ± 5.9                                                                         15.941 ± 2.1 19%                                        __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    SUPPRESSION OF LINE CELLS USING SPLENOCYTES                                   FROM CONTROL OR AEROSOL TREATED RATS AS MODULATORS                                     CONTROL RATS                                                                            AEROSOL TREATED RATS                                                                          %                                          RESPONDERS                                                                             (Δ CMP × 10.sup.3)                                                          (Δ CMP × 10.sup.3)                                                                SUPPRESSION                                __________________________________________________________________________    NLB + CON A                                                                            93.404 ± 1.4                                                                         87.635 ± 8.1 6%                                         NLB + GP-MBP                                                                           63.023 ± 12.9                                                                        35.523 ± 8.9 44%                                        NLA + CON A                                                                            19.892 ± 4.6                                                                         30.553 ± 3.6 -53%                                       __________________________________________________________________________

In Tables 2 and 3, it can be seen that when GP-MBP was added to the NLBline in the presence of modulator cells isolated from the spleen ofaerosol-treated (with GP-MBP) rats, a 44% suppression of the immuneresponse was observed (Table 3). Therefore, MBP was required to suppressthe immune response in aerosol-treated animals and the suppression wasantigen specific. When Con A was added to NLB or NLA cells alone, it didnot induce suppression.

The data in Table 2 shows that thymocytes were not involved in thisimmune suppression as neither the control (NLB+Con A) or MBP-treatedlines showed evidence of any suppression. Therefore, one of themechanisms of action of the aerosolization of autoantigens for thetreatment of autoimmune diseases appears to be the generation of cellsthat actively suppress the specific in vitro autoimmune response intreated animals. This was also demonstrated by adoptive transfer ofprotection by spleen cells from aerosol-treated animal as shown inexample 9 below.

EXAMPLE 4

Adjuvant arthritis is a model for rheumatoid arthritis in whicharthritis is induced by injecting Freund's complete adjuvant into thebase of a rat's tail. Approximately 14 days post-immunization, animalstreated in such a fashion develop severe swelling of their joints,characteristic of rheumatoid arthritis in humans.

The effect of aerosol administration of collagen on adjuvant arthritiswas studied. The experiments were performed as follows.

Aerosolization of Type II collagen was performed as in Example 2 abovefor GP-MBP at doses of 0.003, 0.03 and 0.3, milligram per animal.Arthritis was induced by injecting rats in the base of the tail withFreund's complete adjuvant. Five animals were used for each experimentalgroup as described above in Example 2. The animals were treated on days-5, -3, 0, +2 and +4 relative to the induction of arthritis in theaerosol-treated rats.

The mean arthritis score was graded on a scale of 0-4 for each of fourpaws in the rats as described in J. Exp. Med. 146: 857, 1977 as follows:0-normal, 1-redness only, 2-redness plus mild swelling, 3-severeswelling, 4-joint deformity. The mean arthritis score was calculated oneach day for each group of animals. Individual scores for each paw wereadded together and scores determined for each animal in the group. Themean score was determined by dividing the total score for the group bythe number of animals tested.

As an example, the calculation of a mean arthritis score is set forthbelow.

    ______________________________________                                                                 Total                                                Animal Number                                                                            Score for Each Paw                                                                          Score for each animal                                ______________________________________                                        1          1, 1, 1, 1    4                                                    2          2, 2, 2, 2    8                                                    3          3, 3, 3, 3    12                                                   4          4, 4, 4, 4    16                                                   ______________________________________                                    

The mean arthritis score for the four animals in this group would be:##EQU1##

The results of the above experiments are shown in FIG. 7 in which thex-axis is the days post-immunization and the y-axis is the meanarthritis score.

As shown in FIG. 7, aerosol administration of collagen at 0.03 and 0.3but not 0.003 milligrams per rat significantly decreased the meanarthritis score. Therefore, as in the case of aerosol administration ofMBP for treating EAE administration of collagen to animals afflictedwith adjuvant arthritis was effective in a dose-dependent manner.

EXAMPLE 5

Bovine myelin and bovine MBP were tested for their ability to suppressEAE when administered orally and in aerosol form. Bovine myelin waspurchased from Biopure (Boston, Mass.) and consisted of isolated myelinpurified on a sucrose gradient according to routine procedureswell-known in the art. EAE was induced and Bovine myelin and MBP wereadministered in oral or aerosol form as in Example 2 above. The resultsare set forth in Table 4 below.

                  TABLE 4                                                         ______________________________________                                                    Days PI                                                           GROUP       12      13    14  15  16  17  18  19  20                          ______________________________________                                        Control     0.9.sup.1                                                                             1.7   2.3 2.4 1.9 1.5 0.9 0.7 0.2                         Bovine myeline,                                                                           0       0     0.3 0.4 0.3 0.2 0   0   0                           1000 mg                                                                       Bovine myelin,                                                                            0.3     0.4   0.6 0.8 0.5 0.5 0.1 0   0                           100 mg                                                                        Bovine myelin,                                                                            0.2     0.2   0.5 0.7 0.4 0.4 0.3 0   0                           MBP, 10 mg                                                                    Bovine myelin,                                                                            0       0     0   0.4 0.7 0.5 0.7 0.2 0                           10 mg (A)                                                                     Bovine myelin,                                                                            0.8     0.7   0.8 1.3 1.1 0.7 0.4 0.2 0                           1 mg                                                                          Bovine myelin,                                                                            0.7     1.1   1.3 1.2 1.3 0.9 0.4 0.3 0.1                         0.1 mg                                                                        Bovine-MBP, 0.2     0.4   0.7 0.6 0.4 0.2 0   0   0                           1 mg                                                                          GP-MBP, 1 mg                                                                              0       0     0   0.1 0.3 0.4 0.1 0   0                           ______________________________________                                         .sup.1 Mean paralytic score                                                   A = aerosol                                                                   PI = postimmunization days                                               

Referring to Table 4 above, Bovine myelin was effective in treating EAEwhen administered in oral and aerosol form. Ten mg of Bovine myelinadministered in aerosol form was as effective in suppressing autoimmunedisease symptoms as 1000 mg of the same material administered orally inliquid form. In addition, oral administration of Bovine MBP was alsoeffective in suppressing EAE symptoms induced by GP-MBP.

EXAMPLE 6

Delay-type hypersensitivity (DTH) responses were measured in Lewis ratsin which EAE had been induced and treated with either oral oraerosol-administered GP-MBP. Delay-type hypersensitivity (DTH) is aclassic measure of in vivo cell mediated immunity.

Rats (5 per group) were treated with 5 mg of GP-MBP administered eitherorally or in aerosol form and DTH responses were measured as above.Delay-type hypersensitivity was induced by injecting 10 micrograms ofGP-MBP into the pinna of a rat's ear and the subsequent swellingmeasured 48 hours later according to methods described in J. Immunol.125: 283, 1980.

DTH responses in rats in which EAE had been induced as in Example 2above were measured as above after various treatments. Rats (5 pergroup) were treated with 5 mg/animal of various MBP preparations inaerosol form and DTH responses measured. The results are set forth belowin Table 5.

                  TABLE 5                                                         ______________________________________                                        DTH Response (× 10.sup.-2 Inches)                                       Treatment       Control  Treated  Significance.sup.1                          ______________________________________                                        GP-MBP, -3      1.150    0.180    0.000                                       GP-MBP, +7, +9, +11                                                                           1.150    0.580    0.004                                       GP-myelin       1.150    0.040    0.000                                       GP-MBP fragment (16-35)                                                                       1.150    0.850    0.036                                       GP-MBP fragment (51-70)                                                                       1.150    1.000    0.251.sup.2                                 GP-MBP fragment (121-138)                                                                     1.150    0.911    0.096.sup.2                                 Bovine MBP      1.150    0.330    0.000                                       Rat MBP         1.150    1.080    0.585.sup.2                                 ______________________________________                                         .sup.1 Determined by student's ttest                                          .sup.2 Not significant by student's ttest                                

Referring to the data in Table 5, aerosol administration of GP-MBP 3days before (-3) or on days 7, 9, and 11 post-induction (+7, +9, +11) ofEAE significantly suppressed DTH responses in aerosol-treated rats.Aerosol treatment using whole guinea pig myelin, administered on days-10, -7, -5, -3, 0, +2 and +4 was also effective in suppressing DTHresponses in treated animals.

Certain fragments of GP-MBP (administration as above) were alsoeffective in suppressing DTH responses in aerosol-treated animals. AGP-MBP fragment, corresponding to GP-MBP amino acid residues 16-35,suppressed DTH responses when administered in aerosol form (Table 5); afragment corresponding to GP-MBP amino acid residues 51-70 wasineffective in suppressing DTH responses and a fragment corresponding toGP-MBP amino acid residues 121-138 induced a low level of suppression ofDTH responses. This data shows that certain fragments of GP-MBP wereeffective in suppressing DTH responses.

Finally, aerosolized Bovine MBP was effective in suppressing DTHresponses in animals immunized with GP-MBP whereas aerosolized rat MBPwas ineffective in this-respect. These data also show thataerosolization of a protein itself is not responsible for the immunesuppression of DTH and only specific proteins produced this effect.

EXAMPLE 7

The effects of aerosol administration of GP-MBP on antibody productionin rats in which EAE had been induced was examined.

Antibody responses were measured in animals treated with aerosolizedGP-MBP and a variety of control antigens including histone and bovineserum albumin (BSA). In these experiments, serum was taken and antibodytiters were measured in 5 animals per group treated on days -10, -7, -5,-3, 0, +2 and +4 relative to immunization with GP-MBP in Freund'scomplete adjuvant three weeks after immunization in the treated rats.Antibody titers were measured by ELISA (as described in Lider, et, al J.Immunol 142:748-752 1989). The results are set forth in Table 6 below.

                  TABLE 6                                                         ______________________________________                                        SERUM ANTIBODY LEVELS                                                         Group        1/10,000 1/20,000 1/40,000                                                                             1/80,000                                ______________________________________                                        Control      0.520    0.239    0.382  0.143                                   GP-MBP 5 mg (A)                                                                            0.276    0.121    0.089  0.074                                   GP-MBP, 1 mg (A)                                                                           0.579    0.380    0.349  0.250                                   GP-MBP, 1 mg (O)                                                                           0.437    0.275    0.203  0.157                                   Histone, 5 mg                                                                              1.405    1.229    0.782  0.644                                   BSA, 5 mg    0.405    0.489    0.382  0.315                                   PBS          0.913    0.953    0.769  0.624                                   BP-MBP-fragment                                                                            0.226    0.243    0.192  0.132                                   (21-40, 0.174 mg/rat)                                                         ______________________________________                                    

The results presented above show that in animals treated with 5 mg ofaerosolized GP-MBP there was a statistically significant decrease inantibody levels as compared to controls. Neither PBS, bovine serumalbumin (BSA, Boehring-Mannheim) nor histone (Sigma Chemical Co., St.Louis, Md.) suppressed antibody responses. In addition, a GP-MBPfragment corresponding to GP-MBP amino acid residues 21-40 alsosuppressed specific MBP antibody responses in a statisticallysignificant manner when administered in aerosol form. The resultspresented above show a decrease in antibody response to MBP in animalstreated with 5 mg aerosolized MBP.

EXAMPLE 8

EAE was induced in rats and the animals were treated with GP-MBP byaerosol as described above in Example 2 (25 mg of GP-MBP in 5 ml of PBSadministered to five rats per group on days -10, -7, -5, -3, 0, +2 and+4). Control animals were treated with PBS alone. Rats were sacrificed16 days after immunization, their brains fixed in formaldehyde, slidesof paraffin-embedded tissue prepared for individual rats and the numberof parenchymal inflammatory foci in the rat's brains were counted in ablinded fashion. Histologic analysis was performed on the rats asdescribed (Higgins et al., J. Immunol. 140: 440, 1988). The results ofthis. biological analysis is set forth below.

In these studies, there was an absence of cells infiltrating brainparenchyma in MBP-treated animals. The results were as follows:

controls (n=6) #foci=48,25,14,11,8,4; average foci/animal=18.3

aerosol treated (n=4) #foci=2,0,0,0; average foci/animal=0.5

This demonstrated that aerosol treatment not only affected clinicaldisease and immunologic function, but also prevented movement of cellsinto the brain. Movement of cells into the brain is the primarymechanism underlying the disease process in EAE.

EXAMPLE 9

A major question related to the protection of animals from EAE followingaerosolization of GP-MBP is the mechanism of action of the specificimmune suppression. The experiments described below show that activecellular mechanisms are triggered by aerosol administration of MBP. Inthese experiments cells that adoptively transfer protection against EAEto naive animals were recovered from the spleens of aerosol-treatedanimals.

Lewis rats were treated as described above in Example 2 using 5 rats pergroup (5 mg per rat of GP-MBP in PBS administered 7 times). Three daysfollowing the last treatment, spleen cells were removed from treatedanimals, single cell suspensions prepared, injected intraperitoneallyinto 5 naive animals (9×10⁸ cells per each recipient) that were thenimmunized with MBP in Freund's complete adjuvant. The results are shownin FIG. 8.

As shown in FIG. 8, there was a marked diminution of disease severity inrats which received spleen cells from MBP-treated animals. In addition,DTH responses were measured in recipient animals and there was markedinhibition of DTH responses to MBP in aerosol-treated rats (data notshown).

Therefore, spleen cells from aerosol-treated animals were capable ofadoptively transferring protection to the naive recipients against EAEand also downregulated DTH responses in these animals.

What is claimed is:
 1. A method for suppressing an autoimmune responseassociated with a cell-mediated autoimmune disease in a mammal, whichcomprises administering to said mammal by inhalation an effective amountfor suppressing said autoimmune response of a composition consistingessentially of at least one agent selected from the group consisting ofan autoantigen specific for said disease, and an autoimmune responsesuppressive fragment of said autoantigen.
 2. The method of claim 1wherein said autoantigen is myelin basic protein.
 3. The method of claim1 wherein said disease is multiple sclerosis.
 4. The method of claim 1wherein said autoantigen is type II collagen.
 5. The method of claim 1wherein said autoimmune disease comprises rheumatoid arthritis.
 6. Amethod for suppressing an autoimmune response associated with theautoimmune disease multiple sclerosis in a mammal, which comprisesadministering to said mammal via the pulmonary tract an effective amountto suppress said autoimmune response of at least one member selectedfrom the group consisting of an autoantigen specific for said autoimmunedisease, and an autoimmune disease suppressive fragment of saidautoantigen.
 7. The method of claim 6 wherein said autoantigen is myelinbasic protein.
 8. A method for suppressing an autoimmune responseassociated with autoimmune arthritis in a mammal, which comprisesadministering to said mammal via the pulmonary tract an effective amountto suppress said autoimmune response of at least one member selectedfrom the group consisting of an autoantigen specific for said autoimmunearthritis, and an autoimmune disease suppressive fragment of saidautoantigen.
 9. The method of claim 8 wherein said autoantigen is typeII collagen.
 10. A method for suppressing an autoimmune responseassociated with a cell-mediated autoimmune disease in a mammal, whichcomprises discharging into the pulmonary tract of said mammal aneffective amount to suppress said autoimmune response of a compositionconsisting essentially of at least one member selected from the groupconsisting of an autoantigen specific for said autoimmune disease, andan autoimmune response suppressive fragment of said autoantigen.
 11. Themethod of claim 10 wherein said autoantigen is myelin basic protein. 12.The method of claim 10 wherein said disease is multiple sclerosis. 13.The method of claim 10 wherein said autoantigen comprises type IIcollagen.
 14. The method of claim 10 wherein said disease comprisesrheumatoid arthritis.
 15. The method of claim 1 wherein said agent is inaerosol form.
 16. The method of claim 6 wherein said agent is in aerosolform.
 17. The method of claim 8 wherein said agent is in aerosol form.18. The method of claim 10 wherein said agent is in aerosol form.